Dipeptides and process

ABSTRACT

Synthesis of phenylserine ester (a) via benzaldehyde and glycine ester using serine hydroxymethyltransferase; and (b) via methyl benzoylacetate. Synthesis of hydroxy-aspertame or derivative by enzymatic coupling of phenylserine or derivative with aspertic acid or derivative. Hydrogenation of the coupled product to give as final product aspertame or analog with related processes and products.

FIELD OF THE INVENTION

In its broad aspect the invention involves the preparation of dipeptidesand their intermediates, many of which are new compounds. The dipeptidesynthesis is accomplished with an effective enzyme and utilizes as oneof the reactants a novel group of compounds analogous to phenylserine.Synthesis routes for the latter group are given.

In a preferred embodiment the invention involves the preparation of(2S,3S)-beta-phenylserine ester and the condensation of the ester withblocked (S)-aspartic acid to make hydroxy-aspartame (a new compound,useful as a sweetener) and certain aspartame homologs and analogs.Several basic stages are involved, each of which has more than one step.Various compositions are made during the course of the overallprocesses.

For many years aspartame has been produced commercially by couplingZ-aspartic acid with phenylalanine ester. However, phenylalanine is madeby fermentation or complex chemical/enzymatic processes and isexpensive. Efforts to find a cheaper aspartame process have been longcontinuing, but, prior to the instant invention, have been unsuccessful.The novel processes herein described are believed to present a cheaperroute.

ABBREVIATIONS AND DEFINITIONS

The following are conventional in this art and are used from time totime herein:

Q means a blocking group, e.g., Z (which is carbobenzoxy), as used toblock the amine group on L-aspartic acid. Q is further defined in aseparate section below.

Me means methyl.

Ph means phenyl.

Phe means phenylalanine.

Z means carbobenzoxy.

Asp means aspartic.

Ser means serine.

APM means aspartame.

BOC (or Boc) means t-butoxycarbonyl.

Lower alkyl means alkyl having 1 to 4 carbons inclusive unless otherwisestated.

Ts means tosyl, i.e., teluenesulfonyl.

DMSO means dimethylsulfoxide.

Metallo-proteinase means proteolytic enzyme having a metal ion at theactive center. Examples are those originating from microorganisms suchas a neutral protease, originating from actinomycetes and includeprolysin, thermolysin, collagenase, crotulus atrox protease; thoseproduced from microorganisms such as Bacillus subtilis, Bacillusthermoproteoliticus, Streptomyces coespitosus, Bacillus megaterium,Bacillus polymyxa, Streptomyces griseus, Streptomyces naraensis,Streptomyces fradiae, Pseudomonas aeruginosa, Asperillus oryzae,Clostridium histolyticum, Proteus aeruginosa, Aeromonas proteolytica,and the like. Crude forms are included. For example, the termthermolysin includes crude thermolysin. Other useful enzymes aretrypsin, papain, and pronase.

THE Q GROUP

In the synthesis of certain peptides and polypeptides, measures must betaken to prevent the amine group of a given amino acid from reactingwith the carboxylic group of another molecule of the same compound (orindeed with the identical molecule). To prevent this, at least one ofthe reactive radicals has to be blocked. Generally, the amine group onone reactant is chosen, leaving a carboxyl free to react with a freeamine group on the other reactant. In the instant case, the amine groupon aspartic acid is blocked, leaving adjacent --COOH free to react withamine on phenylserine ester.

Such blocking (or masking or protecting) groups are herein referred toas "Q" groups.

The expedient of the blocking group was conceived by Emil Fischer in thecourse of his polypeptide work during the early years of this century.Fischer explored the use of a great many amine blocking groups, and alarge number of additional groups have since been suggested. Many ofthese N-substituents result in a urethane-type group, attached to N,e.g., --NH--C(:O)OR, where R can be alkyl or indeed substantiallyanything that completes a urethane group. These materials are well knownto those skilled in the peptide art. Typical Q radicals are carbobenzoxy(known as benzyloxycarbonyl), p-methoxybenzoxycarbonyl,t-butoxycarbonyl, etc. At the end of the synthesis, Fischer removed theQ group by a simple procedure and peptide syntheses have proceded inanalogous manner since Fischer's time. Q removal by specifichydrogenation or the like is an intermediate step in the instantinvention and in fact results in formation of a new compound.

In the dipeptide art carbobenzoxy, C₆ H₅ CH₂ OCO--, is so frequentlyused as a blocking group that it is referred to simply as "Z". It isespecially convenient to use in that the group as the acid chloride(carbobenzoxychloride) reacts readily with the amine group in aSchotten/Baumann synthesis, and yet is readily eliminated from thepeptide by catalytic hydrogenation, as toluene and carbon dioxide. Otherblocking Q groups are readily removable by means well known to thoseskilled in the dipeptide art. For example, BOC is easily removed bytreatment with acid. (See example 10.)

In general terms Q includes tertiary alkoxycarbonyl groups such ast-butoxycarbonyl (already mentioned), phenylacetyl, acetoacetyl,N-benzylidene, benzoyl, benzyl, t-amyloxycarbonyl; benzyloxycarbonylgroups (including Z, already mentioned), p-methoxybenzyloxycarbonyl;3,5-dimethoxybenzyloxycarbonyl; 2,4,6-trimethylbenzyloxycarbonyl;p-phenylazobenzyloxycarbonyl; p-toluenesulfonyl; o-nitrophenylsulfonyl;trifluoroacetyl; chloroacetyl; carbamyl; and the like.

Thus Q refers to a conventional group, easily reacted with the aminogroup of the relevant amino acid, blocking further reaction of thatamine group with carboxyl in peptide synthesis, and yet readily removedwhen the peptide snythesis is over. Accordingly, Q terminology is usedin this classical sense, and is not to be construed as limiting therelevant reactants to specific chemically-defined substituents.

SUMMARY OF THE INVENTION

In a major aspect the invention is directed to a process for producing adipeptide having the formula ##STR1## wherein B represents hydrogen orQ; Q represents an amino acid protective group; V represents hydrogen oran alkyl group having 1, 2, 3, or 4 carbon atoms; C¹ and C² have thecommon natural configuration of naturally occurring amino acids; and

(a) Y is hydrogen and X is HET, where HET represents a hetero-atomic orsubstituted hetero-atomic group releasable from carbon by reductivecleavage; or

(b) each of Y and X is HET; or

(c) X and Y and C³ together comprise a HET ring or a non-cyclic HETgroup;

said process comprising:

reacting (i.e., coupling) B-substituted aspartic acid of the formula##STR2## or a salt (e.g. an acid or an amine salt) thereof with a secondamino acid having the formula ##STR3## wherein X, Y, and V are asdefined above and the C² carbon has the common natural configuration ofnaturally occurring amino acids;

said reaction being conducted in

(d) a water-immiscible solvent in the presence of a metallo-propteinase,

(e) a water-miscible solvent in the presence of a nonmetallo-proteinase,or

(f) a water-immiscible solvent in the presence of anon-metalloproteinase.

The above solvent systems include biphasic or multiphase systems (e.g.,a solid bound enzyme phase plus water-immiscible organic and aqueousphases), where the enzyme and substrates are dissolved in the aqueoussolution and the dipeptide product diffuses to the organic phase.

Certain substituent groups in Formulas I, II, and III are preferred,viz., where:

(a) Y is hydrogen and X is --OR', --SR', --OC(:O)R', --OC(:O)OR,--OC(:O)NHR', --OC(:S)SR, --Cl, --Br, --N₃, --OS(:O)(:))--R,--S(:O)(:O)--R, --NHR', or --NO₂ ; or

(b) X and Y are independently --OR', --SR', RS(:O)(:O)--, --OC(:O)R',--NHR', or --Cl; or

(c) Y and X together are ═O, --S(CH₂)_(n) S--, --S(CH₂)_(n) O--,--O(CH₂)_(n) O--, ═NNHC(:O)NH₂, ═NNHC(:O)R', RNHN═, TsNHN═, or ═NOH; and

R' is H or R; R is alkyl or alkylene having 1, 2, 3, or 4 carbons, aryl,or substituted aryl; n is 1, 2, 3, or 4; and B is Q.

"Substituted aryl" includes groups such as aralkyl, alkaryl,aliphatic-substituted aryls, and the like. "Alkyl" and "alkylene"include substituted alkyls and substituted alkylenes. X and Y areinterchangeable.

There are even further substituent preferences, e.g., where:

(a) Y is H and X is --OR', --SR', --OC(:O)R', --OC(:O)OR, --OC(:O)NHR',--OC(:S)SR, or --NHR'; or

(b) X and Y are independently --OR' or --SR'; or

(c) X and Y together are --S(CH₂)_(n) S--, --O(CH₂)_(n) O--, TsNHN═,═NNHC(:O)NH₂, ═NNHC(:O)R', or ═NOH.

Of (a) (b), and (c) immediately above, further sub-groups are preferred:

(a) Y is H and X is --OH, --NH₂, or --NHR where R is lower alkyl; or

(b) X and Y are independently --OR'; or

(c) X and Y together are --O(CH₂)_(n) O--, TsNHN═, ═NNHC(:O)NH₂,═NNHC(:O)R', or ═NOH.

Thus as species, Formula III would include, without limitation,

(a) where Y is H, X is --OH, --Cl, --OCOOPh, --OCOOEt, --OCOOMe, --OMe,--OCH₂ Ph, --OC(:O)Me, --OC(:O)Ph, --OC(:O)CH₂ CH₂ CH₃, --OCH₂ CH₃,--OCH₂ CH═CH₂, --OC(:O)NHCH₃, --OC(:S)SMe, --OC(:S)SEt, --SMe, --SEt,--SPh, and --OC(:S)S--CH₂ CH═CH₂ ; and

(b) X and Y are independently --OMe or --OEt; and

(c) X and Y together form --S(CH₂ S--, --S(CH₂)₃ S--, or ═NNHC(:O)NH₂.

In one aspect the compositions of Formula III are treated reductively(e.g., by catalytic hydrogenation) prior to coupling. Such reducedproducts include phenylalanine and phenylserine, especially where theprecursor is benzoylglycine.

In the text and claims, "HET" is used to define hetero-atomic orsubstituted hetero-atomic groups, including groups in ring form (e.g.,with C³ forming part of the ring). These terms, which apply to the C³carbon in compounds described herein, are used in the conventional senseto means that the immediate substituent is an atom or group other thancarbon. For example, a hetero-atom could be --Cl or --Br, and obviouslythe halogen would be attached only to C³. If the hetero atom ispolyvalent (e.g., oxygen, sulfur, or nitrogen), it would normally bridgeC³ and some other atom or group, e.g., as in the structure HC³ --O-loweralkyl, HC^(3--S--Ph), or --C³ --NH--Ph. The phenyl (or other aryl) groupcan be substituted, e.g., as in tosyl--, HC³ --O--SO₂ C₆ H₅ CH₃. Thehetero-atom can also be oxygen alone, as ##STR4##

In lieu of the hydrogen substituent per the above, both the relevantvalences from X and Y on C³ may be satisfied by hetero-atomic orsubstituted hetero-atomic, e.g., MeO--C³ --OMe, MeS--C³ --SMe, and soon.

Certain polyvalent groups may form a ring with C³. Examples are:##STR5## and so on.

With certain groups the C³ attachment is not a ring but a double bond,e.g., C³ ═NOH, C³ ═N--NH-tosyl, C³ ═N--NH--C(:O)NH₂, C³ ═O, and thelike.

All of the X:Y groups or atoms are releasable from C³ by one or more ofthe known methods of cleavage, either befor or after peptide coupling.By this is meant, the X:Y compound can be treated in such a way that theatoms or groups are replaceable by hydrogen, leaving C³ as a methylenegroup, --CH₂ --. Various methods of reductive cleavage of benzylicsubstituents are well known to those skilled in the art. The exactnature of the reductive cleavage is dictated by the X:Y atoms or groupspresent, and the reduction may result in the direct formation of amethylene or may pass through a stable intermediate which isconcurrently or subsequently removed, such as C═N--OH→C--NH₂ →--CH₂ --.This property is common when X:Y comprise with C³ a multiply-boundoxygen or nitrogen atom. Catalytic hydrogenation is one of the commonesttypes of reductive cleavage of benzylic functional groups. In fact inthis invention all X:Y groups or atoms are replaceable by catalyticmethods. For example HC³ OMe may be reacted with hydrogen over Pd, Pt,or Raney Ni, and the methoxy group will be replaced by hydrogen. (For aleading reference in this area see Khan, A. M., et al, Tet. Let. No. 24,pp. 2649-2655 (1966).)

In addition to catalytic hydrogenation certain of the X:Y groups may bereduced by chemical reducing agents to methylenes. For example, halogenor sulfonic acid ester substituents are reduced to methylenes byreaction with NaBH₄, NaBH₃ CN, Zn in acetic acid, MgH₂, or n-Bu₃ SnH, aswell as other chemical reducing agents known to those skilled in theart. The only consideration to be made in the choice of a reducing agentis its compatibility with other functional groups in the compound to bereduced. Numerous examples of these reductive procedures are containedin the five-volume reference series The Compendium of Organic SyntheticMethods, Ed. Vol. 1 and 2, Harrison and Harrison; Vol. 3, Hegedus andWade; Vol. 4 and 5, Wade; Wiley-Interscience, New York, N.Y.

As used from time to time herein, the language "treating Q, X, and/or Ygroups to replace same with hydrogen" (or equivalent language), is usedbroadly to encompass processes for removing one or more of such groupsand replacing same with hydrogen. As explained in this section, avariety of treatments is available to accomplish this, e.g., acidhydrolysis and various types of reductive cleavage, including catalytichydrogenation; enzymatic cleavage, chemical reduction, and so on. Insome cases different treatments are contemplated for each of the threegroups. Certain groups respond preferentially to certain treatments, andguidelines for typical preferences are provided.

In Formula IV (q.v.), where R¹ =R³, and in the case where the compoundis treated to replace Q with H by acid hydrolysis, the replacementprocess is not strictly reductive cleavage, but still falls within thegeneral language, "treatment to replace with hydrogen."

Acid hydrolysis is recommended for removal of the following Q groups:t-butoxycarbonyl, phenylacetyl, trifluoroacetyl, acetoacetyl, benzoyl,and t-amyloxycarbonyl. Such treatment results in replacing the groupwith hydrogen.

Certain Q groups can be removed enzymatically, e.g., phenylacetyl,chloroacetyl, and carbamyl. In such treatment (whereby the group isreplaced with hydrogen), procedures well-known in the art may befollowed.

In carrying out removal of X, Y, and Q groups (and their respectivereplacement with hydrogen), addition of acid (e.g., a strong mineralacid) is desirable to depress diketopiperazine formation and toaccelerate the rate of removal. The formation of salts facilitates thetreatment to replace with hydrogen.

In the basic coupling process, an aspartic acid compound (an amino acid)is reacted with a phenylserine compound (a second amino acid). Bothcompounds have asymmetric carbon atoms (respectively C¹ and C² in theabove formulas), and as such may occur naturally, or may be derivativesof, homologs or analogs of, or otherwise share structural similaritieswith naturally occurring compounds, where the natural configuration ofthe amino acids is that given by the Fischer projection, ##STR6## whereR is any group that completes the amino acid.

General methods for making the compositions of Formula III are known tothose skilled in the art. Typical are the reaction of chlorides withN-protected phenylserine esters, e.g., ##STR7## and the like.

Sulfur analogs may require several steps: ##STR8##

Other modes of synthesis are described in the examples. See especiallyExamples 14-22.

To complete the process of the invention the dipeptide of Formula I istreated to form a reduced derivative (which may be aspartame); which isto say, ##STR9## wherein R³ =R¹ or R², and ##STR10## is treatedreductively (e.g., by hydrogenation or the like) to form a compound ofthe formula ##STR11## wherein at least one of X' and Y' representsrespectively X and/or Y as above defined, replaced by hydrogen as aresult of the reductive treatment. Where R³ is R², X' and Y' are bothhydrogen, and V is methyl the compound is aspartame,L-alpha-aspartyl-L-phenylalanine methyl ester, ##STR12##

In the aforesaid reductive process (going from Formula I to Formula IV)new compounds are formed, viz., ##STR13## in which R³, X', and Y' are asabove defined, except that X'--C*--Y' excludes CH₂.

Formula VI differs from Formula IV in that Formula VI represents a classof new compounds, and so may not include aspartame, a known compound,which would result when R³ =R² and both X' and Y' are H; henceX'--C*--Y' excludes CH₂.

BACKGROUND OF THE INVENTION WITH A CONSIDERATION OF CERTAIN PRIOR ART

It is known to make aspartame by enzymatic coupling of blocked asparticacid with phenylalanine methyl ester. While the mechanism is undoubtedlycomplex, the overall result is a simple dehydration, thus: ##STR14##

The amine on aspartic acid is blocked with carbobenzoxy (i.e., a "Q"group) to prevent undesirable side reactions.

Aspartame is useful as a sweetener only in the L,L-form, i.e., when its2 chiral carbons (in this instance one in the phenylalanine moiety andone in the aspartic acid moiety) are in the L form. The 3 otheroptically-active isomers (L,D; D,L; and D,D) are bitter or tasteless.Procedures using phenylalanine are known that will give the L,L-form,substantially free of undesirable isomers.

U.S. Pat. No. 4,284,721, Oyama et al, discloses the foregoing reactionto give the L,L-form, using various immobilized enzymes, includingthermolysin. The pores of the immobilized enzyme matrix are filled withwater, and thus the reaction of aspartic acid and phenylalanine iscarried out in water. The 2 reactants are however dissolved in anorganic solvent immiscible with water (e.g., ethyl acetate), and thatsolution contacts the water-containing immobilized enzyme. Yields ofL,L-ZAPM are stated variously as 25.5-88%. The inventors in U.S. Pat.No. 4,284,721 published a parallel paper dealing with the same reaction,reactants, and enzyme, Oyama et al, J. Org. Chem., 1981, 46, 5241-5242,stating,". . . substrates move from the organic layer to the aqueouslayer of the support, where the reaction takes place, and then theproduct diffuses back to the organic layer effectively . . . " Thispaper also mentions that in organic solvents "the reaction rate israther slow as compared with that in aqueous solution." And see Oyama etal, Enzymatic Production of Aspartame, Enzyme Engineering, 7, pp. 96-98,disclosing reaction of L-aspartic acid with D,L-phenylalanine to giveL,L-aspartame, using thermolysin. The reaction is carried out in water.The reaction product is in the form of an "insoluble addition compound",ZAPM.PheOme. (See Isowa below.) The Z group is removed by catalytichydrogenation.

Isowa et al, Tetrahedron Letters, No. 28, pp. 2611-2612 (1979),discloses that the thermolysin-induced reaction of Z-L-aspartic acidwith L-phenylalanine-OMe in water gives Z-L-Asp-L-Phe-OMe.L-Phe-OMe;which is to say, the L,L-reaction product forms an addition product withthe PheOMe reactant. The enzyme was not immobilized. When racemic mixesof reactants were used, only the L,L-aspartame product was precipitatedas the addition compound. The phenylalanine portion was separated by useof aqueous hydrochloric acid and the Z group removed by catalytichydrogenolysis, thereby to give free L,L-aspartame. Yields are high,typically in excess of 90%. Formation of such addition compouds byenzymatic coupling in aqueous media is also described in U.S. Pat. Nos.4,116,768, 4,119,493, 4,165,311, 4,256,836, and 4,436,925.

Petkov et al, Enzyme Peptide Synthesis, Tetrahedron Letters, 25, No. 34,pp. 3751-3754 (1980) teaches reaction of Z-Asp with PheOMe in waterusing thermolysin. With excess PheOMe an addition compound is formed(per Isowa et al supra). Reaction times 3-4 hours give excellent yields(typically in excess of 90%).

To summarize certain of the prior art, the reaction of Z-aspartic acidwith phenylalanine methyl ester, using immobilized thermolysin:

(a) in water, the reaction is fast, with good yield of an additioncompound, Z-L,L-Asp.PheOMe;

(b) in organic solution, the reaction is slower, but no additioncompound separates;

(c) whether in water or organic solution, thermolysin formsL,L-aspartame from racemic reactants, i.e., L,D-Phe+L,D-Asp.

In one step of the instant invention methyl 2-oximino benzoylacetate ishydrogenated to make erytho-beta-phenylserine methyl ester. See Example4. In that connection the following article is of interest.

Elphimoff-Felkin et al, Memoires Presents a La Societe Chimique (1952),pp. 252-264, at p. 259, disclose hydrogenation of ethyl 2-oximinebenzoyl acetate, dissolved in acetic acid, in the presence of PtO₂,using hydrogen. They report a mix of threo and erythro isomers ofphenylserine, stating that the erythro isomer predominated. A repetitionof their work confirms their result, the mix analyzing 75% erythroisomer and 25% threo isomer. The corresponding reaction in the instantinvention differs in use of catalyst (Pd metal, not the French PtO₂) andin the use of solvent (methanol, not the French acetic acid). Thesedifferences result in a yield of essentially pure erythro isomer, andsuch result was not to be predicted. Using the reference Frenchprocedure, 1 g of oxime gives 600 mg of erythro isomer and 200 mg ofthreo isomer, an overall yield of 92% (based on oxime) and an erythroisomer yield of 54.3% based on oxime. This compares with yields of 95+%of pure erythro isomer obtained in the invention process, same basis.See Example 4, using methyl ester, and Example 5, using ethyl ester.

DIFFERENCES OVER THE PRIOR ART

Although phenylserine differs from phenylalanine only by having ahydroxyl group instead of a hydrogen, its enzymatic reaction withaspartic acid is startingly different. As noted, D,L-Phe+L-Asp in water,with enzyme, gives an addition compound; the reaction is fact, the yieldgood. Substitution of D,L-Phe by D,L-erythro-beta-phenylserine methylester, on the contrary, gives a mixture of products, and no additioncompound is separable. See Example 9. The art teaches that the reactionin organic media is slower than in water, and one might expect thatsubstitution of phenylserine for phenylalanine would give results evenworse than phenylserine +aspartic acid in water. It is surprising,therefore, that phenylserine +aspartic acid in organic media not onlygives an excellent yield of Dipeptide I, but proceeds about 2.5 timesfaster than the corresponding reaction using PheOMe+L-Asp.

International Patent Application No. PCT/HU84/00060 filed Dec. 7, 1984discloses hydrogenating a phenylserine derivative of the formula##STR15## to prepare ##STR16## where R is hydrogen or C₁₋₄ alkyl, R² ishydrogen or --C(:O)R⁴, and R⁴ is C₁₋₄ alkyl, aralkyl, or aryl, and R⁵ isH or --CO--R⁴. In several examples D,L-threo-phenylserine ishydrogenated to phenylalanine, and hydrogenation of the erythro-isomeris mentioned.

Tou and Vineyard, J. Org. Chem. 1984, 49, 1135-1136, teach conversion ofthreo-beta-phenyl-L-serine to hydrochloride salt ofthreo-O-acetyl-beta-phenylserine, which is converted by hydrogenolysisto N-acetyl-L-phenylalanine, followed by hydrogenolysis of the latter toL-phenylalanine.

Japanese Pat. No. 7,332,830, WPI Acc. No. 75-31430W/19, of Feb. 28,1979, discloses condensation of glycine and benzaldehyde with threoninealdolase.

PREPARATION OF (2S, 3S)-BETA-PHENYLSERINE ESTER An Overview

Consider the formula of beta-phenylserine: ##STR17##

Four stereoisomers are possible and in fact are known; two in theerythro form, comprising the (2S,3S)-configuration and its mirror image,(2R,3R)-; and two in the threo form, (2R,3S)- and its mirror image(2S,3R)-. Two racemic mixes of these four are also known, i.e., theerythro form, (2RS,3RS)-, and the threo form, (2RS,3SR)-. See Dictionaryof Organic Compounds, pp. 238-239. This use of absolute configurationconforms to the Cahn, Ingold, Prelog convention.

Of the four beta-phenylserine isomers, only the (2S,3S)-form is utilizedby the coupling reaction described in Example 8. This isomer can be usedeither in its pure form, or in the form of the erythro racemic mix,(2SR,3SR)-, or simply (SR,SR), which racemate consists of(2S,3S)-beta-phenylserine and its mirror image,(2R,3R)-beta-phenylserine. Of this racemate (in the ester form) only the(2S,3S)-isomer enters the coupling reaction of Example 8. The reason forthis is that the enzyme is selective and will condense only the(S,S)-isomer under the conditions of Example 8. The same is true of theblocked aspartic acid reactant, Q-Asp. (For abbreviations anddefinitions, see below.) Given the discovery that the S,S-isomer ofphenylserine works, one would expect that the 2S,3R-isomer would alsowork; but, as above noted, it does not. It is surprising, therefore,that the S,S-isomer works, and that it alone of the four optical isomersworks. Indeed, its use is critical. The phenylserine reactant mayinclude the threo-isomer, provided the erythro-isomer is also present.

In Example 8 the beta-phenylserine reactant is the erythro racemate,(2SR,3SR)-, methyl ester, and this preferential product is made by twonovel processes, namely:

(A) Condensing benzaldehyde with glycine methyl, ethyl, or propyl esterto give phenylserine ester, using serinehydroxymethyltransferase--herein referred to as the SHMT Process; and

(B) Condensing methyl benzoate with an alkyl acetate, converting thecondensate to the beta-keto oxime, and reducing the oxime toerythro-phenylserine ester--herein referred to as the Methyl BenzoateProcess.

THE SHMT PROCESS BACKGROUND

It is known to condense glycine with formaldehyde to give L-serine usingserine hydroxymethyltransferase (SHMT) (also known as serinetranshydroxymethylase). See Hamilton et al, Manufacture of L-Amino Acidswith Bioreactors, Trends in Biotechnology, 3, No. 3, pp. 64-68; and U.K.patent application No. 2,130,216A of Nov. 18, 1983. Nakazawa et al, inU.S. Pat. No. 3,871,958, Mar. 18, 1975, disclose the enzymaticcondensation of benzaldeyde with glycine to give beta-phenylserine; SHMTis not specified, and whether it was in fact used in uncertain. U.S.Pat. No. 3,871,958 also teaches condensation of benzaldehyde withethanolamine. So far as can be determined, the reaction of benzaldehydewith a glycine ester using SHMT is novel. In fact, apparently the onlyreference to glycine ester and SHMT in the literature is Ulevitch et al,Biochemistry, 16, No. 24, pp. 5342-5463 (1977), disclosing cleavage ofbeta-phenylserine to benzaldehyde and glycine ester. This reaction is ofcourse the opposite of the invention process.

SHMT is readily available. See Schirch et al, J. Bact., 163, No. 1, pp.1-7 (July 1985); and Ulevitch et al, op. cit.

Considerable confusion exists in the literature and in patentsconcerning the identity of enzymes catalyzing the reaction describedabove. Enzymes have been reported with names such as threonine aldolaseand allothreonine aldolase. D-specific counterpart enzymes have alsobeen reported. At times these activities have been shown to be the sameenzyme, but at other times separate enzymes have been shown to catalyzethese reactions. To add to the confusion mammalian cells possessmitochondrial and cytosolic SHMT activites. These enzymes are clearlydifferent, and furthermore the activity of SHMT towards varioussubstrates varies from one mammalian cell type to another.

SHMT and its relatives have been reported in eucaryotes--fungal, plant,and animal cells--and in procaryotes (bacteria). Most of the informationon SHMT is based on mammalian cell enzyme. Because large quantities ofenzyme are more readily available from a bacterial source, the inventiveprocess has chosen to use SHMT from Escherichia coli (E. coli). The E.coli strains used in the invention as enzyme source were geneticallyengineered to produce elevated levels of SHMT. SHMT is the product ofthe E. coli glyA gene. This gene was inserted into the tetracyclineresistance gene of pBR322, resulting in loss of resistance to thsantibiotic in transformant bacteria. The gene is on a 3.3-kilobase Sa1I-EcoRI fragment; plasmid is designated pGS29. The plasmid codes forresistance to ampicillin allowing for selection of bacteria transformedwith the plasmid. pGS29 was inserted into two E. coli host strains--DH2and HB101.

EXAMPLE 1 Preparation of Beta-phenylserine methyl ester fromBenzaldehyde and Glycine Methyl Ester Using SHMT from E. coli asCatalyst

Cells of DH2/pGS29 grown in complex broth medium were disrupted inphosphate buffer plus pyridoxal-5-phosphate (P-5-P), and this crudeextract was used as the SHMT enzyme source. The extract was added toreaction mixtures containing, at initial concentration, 150 millimolar(mM) glycine methyl ester, 100 mM benzaldehye, and 50 micromolar (mM)P-5-P in phosphate buffer at pH 8.0. Samples were removed from reactionmixtures at 0, 2, and 4 hour intervals of reaction time. The sampleswere analyzed by high performance liquid chromatography (HPLC) forbeta-phenylserine methyl ester. The amount of erythro and threo isomerswas also determined by this method.

After 2 hours of reaction 1.48 g/l of beta-phenylserine methyl ester wasproduced, 83% of which was the erythro isomer. By 4 hours theconcentration of beta-phenylserine methyl ester had increased to 2.14g/1; at this point the erythro isomer represented 82% of the total.

For use in this invention, SHMT requires pyridoxal-5-phosphate (P-5-P),e.g., at 5 mM-5 mM P-5-P per 100 mM benzaldehye.

SOME VARIATIONS IN THE SHMT PROCESS

The concentration of the substrates can vary. An operable range ofconcentration for benzaldehyde is about 10 to 100 mM, with aconcentration of about 100 mM preferred. The concentration of glycineester can be within the range of about 10 to 150 mM. The upper limit isfixed only by the solubility of the ester, which is about 150 mM. It ispreferred that the reaction mixture be saturated with glycine ester.

The SHMT may be immobilzed, using any of a variety of supports andimmobilization techniques well-known to those skilled in the art. In theExample, whole cells, were used, but ths is not necessary.

In the Example, 0.6 units of SHMT per ml were used. The concentrationmay be as low as 0.05 units/ml. (A unit of SHMT is equal to that amountof enzyme which catalyzes production of 1 micromole of benzaldehyde perminute from phenylserine.)

The coupling reaction can be carried out at about 10° to 65° C.,preferably in the range 30° to 40° C. The reaction mixture ahould bemaintained at a pH of about 6.5-9, preferably 7.5-8. The synthesis canbe batch-wise or continuous. In one embodiment the reaction may becarried out in a water-miscible (e.g., methanol) or water-immiscible,organic solvent (e.g. ethyl acetate).

THE METHYL BENZOATE PROCESS

Turning now to the methyl benzoate route for making beta-phenylserine,in summary, (i) methyl benzoate is condensed with lower alkyl acetate(examplified here with methyl acetate) over sodium, forming methylbenzoylacetate and by-product methyl alcohol. (ii) The former is treatedwith sodium nitrite to form the oxime, which is then, (iii) hydrogenatedto form a racemic mixture (1:1 S,S/R,R) of beta-phenylserinestereoisomers; i.e., beta-phenylserine methyl ester as the erythroracemate.

Steps (i) and (ii) are old in the art; (iii) is carried out in a novelway per this invention. The integrated series of step (i), (ii), and(iii) as above stated broadly, is believed novel, as are (ii) and (iii)taken together. Thus the invention includes (i)+(ii); and (ii)+(iii).

Reference is made to the following schema. ##STR18##

The product of Formula VIII is a racemic mix, i.e., (SR,SR)-phenylserinemethyl ester, or erythro-beta-phenylserine methyl ester. This racemateprovides the starting reactant for the next stage of the invention,coupling (S,S)-phenylserine ester with Q-aspartic acid, per Example 8.Experimental details for the preparation of erythro-beta-phenylserinemethyl ester hydrochloride and its precursors follow.

EXAMPLE 2 Preparation of Methyl Benzoylacetate

    PhCOOCH.sub.3 +CH.sub.3 COOCH.sub.3 +Na→PhCOCH.sub.2 COOCH.sub.3 +CH.sub.3 OH

A 1-L flask fitted with a mechanical stirrer, reflux condenser, andnitrogen sweep (to protect the Na), was immersed in a water bath tocontrol temperature (i.e., to provide heat and to cool if need be). Inthe flask was placed 272.3 g (2 moles) of methyl benzoate; 74.1 g (1mole) of methyl acetate; 1 gm. atom--23 g., Na; and 32 g (1 mole) ofmethanol (to react with the Na and to initate the reaction). The flaskwas purged with nitrogen and was maintained under a positive nitrogenpressure throughout the reaction. The solution was heated to 80°-85° C.overnight, during which time all the Na metal was consumed. Theresulting yellow heterogeneous solution was cooled to room temperatureand poured into a separatory funnel containing 130 ml of concentratedhydrochloric acid and 200 g. of crushed ice. This was shaken and thelower aqueous phase removed. (In this step the Na in the Na methylbenzoyl acetate reacts with the HC1 and is removed as NaCl.) Theresidual material was then washed with water, 2×100 mls, saturatedNaHCO₃ solution, 2×100 mls. and finally 2×100 mls of saturated brine(NaCl). (Byproduct methanol leaves with the water in the water washes).The residual yellow organic phase was then transferred to a distillingflask and fractionated through a 12-inch Vigreux column at 0.5 mm Hgpressure. A forerum containing methyl benzoate and methyl acetoacetatewas collected at 37°-42° C. at 0.5 mm Hg. This was followed by afraction of 82.5 methyl benzoyl acetate boiling at 81°-84° C. at 0.5 mmHg. Yield of the pure product methyl benzoyl acetate based on Na was46.3%, as a water white liquid.

EXAMPLE 3 Preparation of Methyl 2-oximino Benzoylacetate

    PhCOCH.sub.2 COOCH.sub.3 +NaNO.sub.2 /HOAc→PhCOC(:NOH)COOCH.sub.3

Apparatus was a 500-ml flask, fitted with a magnetic stirrer and anaddition funnel. In the flask was placed 44.55 g. (250 millimoles) ofpure methyl benzoylacetate and 100 ml of glacial acetic acid. Thissolution was cooled to 10°-12° C. (ice bath) and maintained at thistemperature during the addition of 20 g. (290 millimoles) of NaNO₂dissolved in 35 ml of water. After the addition was complete (30-45minutes) the solution was allowed to warm to room temperature andstirred for an additional 2 hours, during which time white crystalsseparated from the solution. The solution was then poured into 500 ml ofwater and this was filtered. The white filter cake was then washed by2×100 ml of water and dried to give 48.9 g. of the oxime product, m.p.134°-136° C. A sample recrystallized from toluene gave white needles,m.p. 135°-136.5° C.

EXAMPLE 4 Preparation of Brythro-beta-phenylserine Methyl EsterHydrochloride

    PhCOC(:NOH)COOCH.sub.3 +H.sub.2 /catalyst→PhCH(OH)CH(NH.sub.2)COOCH.sub.3

A 500-ml Parr bottle equipped with shaker was used for the reduction. Inthe bottle was placed 20.7 g (100 millimoles) of the oxime, 200 mlmethanol solvent, 15 ml concentrated HCl and, as hydrogenation catalyst,500 mg of 5% Pd (metal) or carbon. The bottle was sealed and degassed invacuo. The shaker was then started and the bottle was maintained at ahydrogen pressure of 15-18 psig by means of a high pressure hydrogentank feeding into a low pressure tank feeding to the Parr bottle, untilhydrogen adsorption ceased (as indicated by a calibrated gauge on thehigh pressure hydrogen tank). Time required, about 1.75-2.5 hours. Thebottle was then vented and degassed in vacuo. Some reaction productsolids came out of solution during the hydrogenation, and the bottle washeated to 60° C. to redissolve such solids. Then the hot solution wasfiltered through a cake of diatomaceous earth (Celite-TM) to removecatalyst. The filtrate was then cooled to 0° C., and glisteningsilver-white platelets separated. This material was collected byfiltration to give 13.6 g of erythro-beta-phenylserine methyl esterhydrochloride product. Concentration of the mother liquor gave another8.2 g of product. Total yield of material was 21.8 g., or 95.2%; m.p.,168°-169° C. ¹ H NMR (400 mHz) (Free base, CDCl₃) δ=1.65 (broad singlet2H); 3.66 (singlet, 3H); 3.78 (doublet, J=5.78, 1H); 4.92 (doublet,J=5.78, 1H); 7.28 (complex multiplet, 5H). In this run, a suitable H₂pressure is 15-60 psig.

EXAMPLE 5 Preparation of Erythro-beta-phenylserine Ethyl EsterHydrochloride

The procedures of Example 3 were followed except that the run startedwith ethyl benzoylacetate, which is commercially available. Thecorresponding oxime, ethyl ester was prepared as in Example 3 in 97%yield, as fine white needles, m.p. 122.5°-123° C. This oxime washydrogenated as in Example 4 to give a 94.9% yield of the phenylserineethyl ester hydrochloride, m.p. 173°-174° C. This was the pure erythroisomer as indicated by NMR. No threo isomer was detected. ¹ H NMR (400mHz) (Free base, CDCl₃) δ=:1.19 (triplet, J=7.16, 3H) 2.0 (broadsinglet); 3.77 (doublet, J=5.70, 1H); 4.10 (doublet of quartets, J=7.16,J_(gem) =4.29, 2H); 4.93 (doublet, J=5.70, 1H); 7.29 (complex multiplet,5H).

These methanol/HCl solutions of substantially pureerythro-beta-phenylserine lower alkyl esters are believed to be novel.They are especially useful as sources of (2S,3S)-beta-phenyl serinelower alkyl esters for reacting with Q-aspartic acid by the process ofthis invention as hereinafter described.

DIPEPTIDE FORMATION An Overview

In the next stage of the invention, (S,S)-beta-phenylserine or an esteror analog as above described is coupled with Q-aspartic acid to form adipeptide, which is then hydrogenated in two steps (or optionally onestep) to form aspartame or an analog as the final product.

Aspartic acid has one chiral carbon, thereby providing two opticallyactive isomers. For use in this invention the L-isomer (S-configuration)is required. A mix of L- and D- (S and R) forms can be used, but the L-(i.e., S-) form will be the effective reactant. Z-L-aspartic acid isavailable commercially and is used preferentially. Unless otherwisestated, "Q-aspartic acid" means the L- (i.e., S-) form.

The coupling process (actually a dehydration) proceeds as follows:

Step 1. Erythro-beta-phenylserine lower alkyl ester (methyl ester isused here as the examplar) is reacted with blocked aspartic acid (with Zas the exemplar block) in organic medium, in the presence ofmetallo-/proteinase as coupling enzyme (immobilized thermolysin is usedas the exemplar), thus: ##STR19## which isN-carbobenzoxy-L-alpha-aspartyl-L-erythro-beta-phenylserine methylester. Using absolute configuration, it isN-carbobenzyloxy-(S)-alpha-aspartyl-(2S,3S)-beta-phenylserine methylester. In the interests of brevity it will be hereinafter referred to onoccasion as "Dipeptide I". Dipeptide I is believed novel, as in theclass of dipeptides comprising it, where Me is lower alkyl generally and--COOCH₂ Ph is Q generally. (See Formula XII below.)

In the next step Dipeptide I is catalytically hydrogenated, using Pd oncharcoal, under relatively mild conditions, thereby removing theblocking group, Z. The resulting compound is ##STR20## which isL-alpha-aspartyl-L-erythro-beta-phenylserine methyl ester, orhydroxy-aspartame. Using absolute configuration, hydroxy-aspartame is(S)-alpha-aspartyl-(2S,3S)-beta-phenylserine methyl ester. It is usefulas a sweetening agent. Hydroxy-aspartame is likewise believed novel. Itis immediately useful in the next step, described as follows:

The hydrogenation is continued, at higher temperatures and pressures,thereby to reduce the -OH group on the phenylserine moiety, givingaspartame, ##STR21##

Aspartame is L-alpha-aspartyl-L-phenylalanine methyl ester.

In one embodiment, the hydrogenation of Dipeptide I is carried throughdirectly to aspartame. Hydroxy-aspartame is an intermediate. See Example13.

Experimental details for the preparation of Dipeptide I will now begiven. This preparation involves making precursors, namely immobilizedthermolysin and its activation; and preparation of the free phenylserineester. Data for making immobilized thermolysin and its activation followthe literature and are given here for the sake of completeness.

EXAMPLE 6 Preparation of Immobilized Thermolysin

A polyacrylate resin (3 g), commercially available as Amberlite XAD-7,was washed on sintered glass with ethanol and with a 25 millimolarTris-HCl buffer, pH 7.5, containing 16 millimolar calcium chloride."Tris" is an abbreviation for tris(hydroxymethylamino methane).Thermolysin, 750 mg, was dissolved in 15 ml ice-cold 25 millimolarTris-HCl buffer containing 16 millimolar calcium chloride and 5 molarsodium bromied, pH 7.5. The washed resin was added to the enzymesolution and the mixture was shaken in the cold room at 4° C. for 17hours. Part of the solution (7.5 ml) was withdrawn and 7.5 ml of 25%glutaraldehyde (crosslinking agent) was added, giving 15 ml of totalsuspension that was shaken at 4° C. for 3 hours. The thus immobilizedenzyme was filtered on a sintered glass and was washed with 0.1 molarTris.HCl, pH 7.5, containing 5 millimolar calcium chloride and 1 molarsodium chloride, and was washed again with the same buffer except notcontaining the sodium chloride. The concentration of immobilized enzymewas 50-80 mg/g of wet resin.

A two-phase liquid was prepared in a separatory funnel comprising 50 mlethyl acetate and 50 ml of 0.1 molar 2(N-morpholino)ethane sulfonic acidat pH 6.0. This mixture was incubated with shaking from time to time for20 minutes. The phases were separated and the immobilized thermolysin (6g) was added to the saturated aqueous phase and the mixture was shakengently at 40° C. for 20 minutes, filtered, and was available for use inExamples 8 and 10. The saturated organic layer was used as the reactionmedium in Examples 8 and 10.

EXAMPLE 7 Preparation of Phenylserine Ester Free Base

The coupling reaction of Examples 8 and 10 requires the free base ofphenylserine lower alkyl ester. Therefore the ester hydrochloride ofExample 4 is neuteralized with base to provide the free ester, asfollows. D,L-Erythro-beta-phenylserine methyl ester hydrochloride, 7 g.,and sodium carbonate, (3.2 g., in 125 ml water, and chloroform, 200 ml,were taken in a 500-ml separatory flask. The two phases were separatedand the aqueous phase was extracted with chloroform, 2×100 ml; theorganic phase was washed with saturated sodium chloride, dried overanhydrous magnesium sulfate, and evaporated to dryness to give acolorless solid of the phenylserine ester, free base. M.p., 100°-101° C.

EXAMPLE 8 Coupling erythro-beta-phenylserine methyl ester withZ-aspartic acid to make Dipeptide I

Immobilized thermolysin prepared as in Example 6 (6 g.) was added to a125-ml Erlenmeyer flask which contained 1.12 g (4.2 millimoles) ofZ-aspartic acid and 2.5 g of D,L-erythro-phenylserine methyl ester (12.8millimoles) in 30 ml saturated ethyl acetate from Example 6. Thereaction mixture was shaken at 40° C. for 8 hours in a mechanicalshaker. The course of the reaction as monitored by high performanceliquid chromatography (HPLC). After 8 hours, according to HPLC, 93% ofZ-aspartic acid was consumed. The immobilized enzyme was filtered offand washed with ethyl acetate. The organic phase was washed with 1 MHCl, 2×20 ml, and water, 20 ml, then dried over magnesium sulfate.Filtration and evaporation afforded an oil, being Dipeptide I, namely,N-carbobenzoxy-L-alpha-aspartyl-L-erythro-beta-phenylserine methylester. (Unreacted phenylserine ester was removed as the hydrochloride inthe HCl wash.)

The Dipeptide I oil was dissolved in a minimum amount of chloroform, andthen hexane was added until the solution became turbid. The Dipeptide Iproduct crystallized from this solution as slightly colored (yellow)crystals. (The color can be removed if desired by treatment withdecolorizing charcoal.) Yield of Dipeptide I, 1.2 g; m.p. 127°-128° C.;[α]_(D) ²⁰ =-7.0° (C=1, methanol).

¹ H NMR (400 M Hz) (δ): 2.61 (doublet of doublet, J_(gem) =17.2 Hz,J_(vic) =5.5 Hz, 1-H), 2.95 (doublet of doublet, J_(gem) =17.2 Hz,J_(vic) =4.4 Hz, 1-H), 3.49 (singlet, 3-H), 4.51 (multiplet, 1-H), 4.83(doublet of doublet, J_(CH-NH) =8.0 Hz, J_(vic) =3.6 Hz, 1-H), 5.02(singlet, 2-H); 5.07 (doublet, J_(vic) =3.6 Hz, 1-H), 6.23 (doublet, J=8Hz, 1-H), 7.1-7.3 (complex, aromatic 10-H).

¹³ C NMR (100.6 MHz)(δ); 36.16 (CH₂), 51.01 (CH), 51.74 (CH₃), 58.90(CH), 66.86 (CH₂), 73.64 (CH), 125.60, 127.40, 127.82, 128.23, 135.77,139.36 (C-H aromatics), 155.81, 169.20, 170.86, and 173.22 (C═O).

Mass Spectrum: 445 [(M+H)⁺ ], 427 [(M+H--H₂ O)⁺ ].

EXAMPLE 9 Attempted coupling of erythro-beta-phenylserine methyl esterwith Z-aspartic acid in water to make Dipeptide I

Z-aspartic acid, 534 mg (2 mmol) and erythro-beta-phenylserine methylester hydrochloride, 926 mg (4 mmol) were dissolved in water, 12 ml, andthe pH was adjusted to 6.2 with 4N NaOH. Thermolysin, 10 ml, was added,and the solution was shaken at 40° C. After 3 hours, some solidprecipitated and after 15 hours 2 phases were obtained. The lower phasewas extracted with ethyl acetate. Analysis by HPLC showed it containedmainly benzaldehyde. The reaction was discontinued owing to thedecomposition of phenylserine into benzaldehyde and glycine Me ester.*

This failure in water to form an insoluble addition product should becompared to and distinguished from results using phenylalanine.

EXAMPLE 10 Synthesis ofN-tert-Butoxycarbonyl-alpha-aspartyl-L-erythrophenylserine methyl ester

The immobilized thermolysin from Example 6 (6 g) was added to a 125 mlErlenmeyer flask containing 0.5 g (2.1 mmol) of N-BOC-L-aspartic acidand 1.22 g (6.3 mmol) of D,L-erythro-phenylserine methyl ester in 20 mlbuffer-saturated ethyl acetate. The reaction mixture was shaken at 40°C. for 10 hours. The product was isolated as described in Example 8.After evaporation of the solvent the product was obtained as a yellowishfoam. ¹ H NMR δ 2.7 (doublet of doublet, J_(gem) =15 Hz, J_(vic) =5 Hz,1-H), 2.8 (doublet of doublet, J_(gem) =15 Hz, J_(vic) =4 Hz, 1-H), 3.5(singlet, 3-H), 3.55 (singlet, 9H), 4.55 (multiplet, 1-H), 4.85(multiplet, 1-H), 5.25 (broad singlet 1-H), and 7.15-7.4 (complex,aromatics).

Mass spectrum: 411[(M+H)⁺ ], 355 [(M+H--C₄ H₈)⁺ ], and 377 [(M+H--C₄ H₁₀O)⁺ ].

EXAMPLE 11 Hydrogenation of Dipeptide I to Make Hydroxy-Aspartame,L-alpha-aspartyl-L-erythro-beta-phenylserine methyl ester

In this operation the Z group on the aspartyl moiety of Dipeptide I isreplaced with H. Dipeptide I(N-carbobenzoxy-L-alpha-aspartyl-L-erythro-beta-phenylserine methylester), 200 mg (0.5 millimoles) is placed in a 15×150 mm test tube. Inthe test tube is placed 2 mls glacial acetic acid, 0.1 ml (1.2millimoles) of concentrated HCl, 80 mg of 20% Pd(OH)₂ on carbon. Thistube was placed in a 500-ml Parr bottle. The bottle was sealed anddegassed in vacuo, then purged with 3×25 psig hydrogen. The purgedsolution was then shaken and pressurized to 45 psig with hydrogen atambient temperature. An aliquot of the solution was analyzed at 2.5hours, and this indicated that less than 2% starting material remained.The solution was then filtered and the catalyst cake washed with 2×1 mlof methanol. The resulting filtrate was then concentrated at ambienttemperature under high vacuum to give a pale yellow foam. This materialwas taken up in 1.5 ml of water to give a cloudy solution, which wasfiltered. The filtrate, a clear pale yellow liquid, pH 2.05, was thenadjusted to pH 5.1 with N NaOH. This was done to "neutralize" the HClsalt of the desired product by bringing it to its isoelectric point. Atthis point the product, hydroxy-aspartame, is an oil. Pure material wasobtained by HPLC; or as very fine needles from water/ethanol at pH 5.

Hydroxy-aspartame readily forms salts, e.g., the trifluoroacetate,hydrochloride, hydrobromide, bisulfate, dihydrogen phosphate, and thelike.

HYDROXY-ASPARTAME SWEETENER

Consumable products containing hydroxy-aspartame are novel, and part ofthis invention. This new dipeptide can be incorporated into consumableproducts in a variety of physical forms, e.g., in powders, tablets,granules, dragees, solutions, suspensions, syrups, emulsions, and thelike. They can be used in combination with suitable non-toxic sweeteningagent carriers such as water, ethanol, sorbitol, glycerol, citric acid,corn oil, peanut oil, soybean oil, sesame oil, propylene glycol, cornsyrup, maple syrup, liquid paraffin, lactose, cellulose, starch,dextrin, and other modified starches; and mono-, di-, and tricalciumphosphate.

Combinations of hydroxy-aspartame with sugar or synthetic sweetenerssuch as saccharin likewise can be incorporated into the consumablematerials in accordance with this invention.

Specific examples of consumable materials containing hydroxy-aspartameare fruits; vegetables; juices; meat products such as ham, bacon, andsausage; egg products; fruit concentrates; powdered beverageconcentrates; gelatins; jams; jellies; preserves; milk products such asice cream, sherbert, and sour cream; syrups such as molasses; corn,wheat, soybean, and rice products such as bread, cereal, pasta and cakemixes; fish; cheese and cheese products; nut meats and nut products;beverages such as coffee, tea; noncarbonated and carbonated soft drinks;beers, wines, and other liquors; confections such as candy andfruit-flavored drops; condiments such as herbs, spices, and seasonings;flavor enhancers such as monosodium glutamate; chewing gum; instantmixes; puddings; and coffee whiteners. Consumable toiletries such asmouthwashes and toothpaste as well as proprietary and nonproprietarypharmaceutical preparations can also be sweetened by hydroxy-aspartame.

The amount of hydroxy-aspartame to be added to the consumable product isthe amount which will provide the degree of sweetness desired. This iseasily determined by taste tests.

The invention also includes the method of adding hydroxy-aspartame tothe consumable products, which is to say, the process of sweetening aconsumable product by incorporating thereinto an effective amount ofhydroxy-aspartame.

Hydroxy-aspartame can be added to consumables over a wide range ofproportions, typically within the range 0.05-3 wt. %. The following listof dosages is provided by way of illustration, and not to state limits.

    ______________________________________                                        Amount of hydroxy-aspartame, Wt %                                                                   Consumable                                              ______________________________________                                        1.3                   Powdered orange                                                               beverage concentrate                                    0.3                   Dietetic syrup                                          2.4                   Milk pudding powder                                                           concentrate                                              0.09                 Preserves                                               0.5                   Bottler's cola syrup                                    0.3                   Gelatin dessert                                                               concentrate                                             ______________________________________                                    

During digestion in the human alimentary tract, hydrox-aspartame and itsester homologs hydrolyze back to the component amino acids, includingphenylserine or its lower alkyl ester homolog, as the case may be. Thusthe metabolism does not involve a phenylalanine intermediate.

The lower alkyl ester homologs of hydroxy-aspartame are also useful assweeteners and can be used in the same manner as hydroxy-aspartame.

When free hydroxy-aspartame is heated it tends to cyclize with formationof diketopiperazine. In subjecting hydroxy-aspartame to variousreactions including replacement of Q groups with hydrogen,hydrogenation, and so on, it is generally desirable to carry out thereaction in the presence of acid. Acid stabilizes the hydroxy-aspartame,forming the corresponding salt, and suppresses formation ofdiketopiperazine. This technique (using HCl) was used in Example 11. InExample 12 the BOC group is removed by acid hydrolysis, usingtrifluoroacetic acid, hydroxy-aspartame being stabilized as thetrifluoroacetate salt. That salt is hydrogenated in methanol-HCl inExample 13.

EXAMPLE 12 Preparation of Hydroxy-aspartame Trifluoroacetate Removal oft-Butoxy Group from t-Butoxy-aspartyl-phenylserine Methyl Ester##STR22##

In a 500-ml flask was placed 504 mg (1.2 mmole) oft-butoxy-aspartyl-phenylserine methyl ester and 5 ml of trifluoroaceticacid. This solution was allowed to stand at room temperature for 1 hour.Analysis by HPLC indicated 100% conversion at that point, and theproduct was isolated by adding 50 ml of ether to the solution andallowing the product to crystallize. This material was collected byfiltration and washed with 10 ml of anhydrous ether and dried in vacuoat 56° C. overnight to give 329.6 mg (64.7% yield) of purehydroxy-aspartame tri-fluoroacetate. M.p. 155°-156.5° C., withdecomposition. [α]_(D) ²² =20.94. (C=1.06 H₂ O). Observedrotation=+0.222°±0.001°.

¹³ C-NMR (100.6 mHz) (D₂ O)δ=35.62; 50.13; 53.79; 59.40; 73.52; 117.20(quartet, J₁₉.sbsb.F-₁₃.sbsb.C =291 Hz); 127.34; 129.62; 139.64; 163.76(quartet, J₁₉.sbsb.F-₁₃.sbsb.C =34.9 Hz); 169.21; 171.98; 173.45. ¹H-NMR (400 mHz)(DMSO·D₆) δ=8.82 (doublet, J=8.0 Hz, 1H); 7.30 (complexmultiplet, 5H); 5.97 (broad singlet, 1H); 4.83 (doublet, J=4.3 Hz, 1H);4.53 (triplet, J=7.5 Hz, 1H); 4.02 (doublet of doublets, J=4.3 Hz, J=8.0Hz, 1H); 3.56 (singlet, 3H); 3.35 (broad singlet, 3 H); 2.71 (twodoublet of doublets, J=7.5 Hz, J=17.3 Hz, 2H).

EXAMPLE 13 Reduction of Hydroxy-aspartame Trifluoroacetate to Aspartame##STR23##

In a 500-ml Parr bottle was placed 10 ml of methanol, 100 mg Pd(OH)₂ oncarbon, and 212.2 mg (0.5 mmole) of alpha-L-Asp-L-erythro-PhSerOMe·CF₃COOH. To this was added 1 ml of N HCl, and the resulting solution wasdegassed and purged with 3×25 psig H₂. The bottle was then vented andthe catalyst filtered from the solution. The solution was thenconcentrated in vacuo to 1 ml and the pH was adjusted to 5.1 with 6 NNaOH. The resulting solution was chilled to 0°-5° C. overnight, and thecrystals of a spartame were collected on a Buchner funnel and washedwith 0.75 ml of absolute ethanol. After drying in vacuo at 56° C. for 5hours the pure white crystals of aspartame had a weight of 139.8 mg.

ONE-STEP HYDROGENATION

As above noted, Dipeptide I (or compounds in the Dipeptide I Class) canbe hydrogenated in one step directly to the end product, therebyremoving in one operation the Q group on the aspartic acid moiety aswell as the hydroxyl group on the phenylserine moiety. The result is thefinal ester product (aspartame, when alkyl is methyl): ##STR24##

EXAMPLE 14 Hydrogenation of Dipeptide I direct to Aspartame

The procedure of Example 11 was followed except that the hydrogenationsolvent chosen was 8.5 methanol. 390.9 mg. (0.88 millimoles) ofDipeptide I was used, with 1.5 ml N HCl (1.5 millimoles). The Parrbottle was pressured to 60 psig of hydrogen and the solution heated to45° C. The reaction time was 6 hours. At the end of the reaction thesuspension was filtered and the methanol was removed under high vacuumat ambient temperture. The residual material was dissolved in 8 ml ofwater and the pH was raised from 1.5 to 5.1 with 6 N NaOH. The resultingsolution was chilled to 0°-5° C. and the aspartame crystallized.Crystals were collected by filtration and washed with 2 ml absoluteethanol. This material was placed in an Abherhalden drying apparatus anddried under high vacuum (0.01 mm Hg) at 56° C. for 24 hours Yield, 220mg., 85%. M.p., 246°- 248°C.

Additional information further explaining the invention follows inExamples 14-22.

EXAMPLE 15 Synthesis of Benzoylglycine

Benzoylglycine, Ph-C(:O)CH(NH₂ COOH, can be used as an intermediate in anumber of the syntheses for making compounds within Formula III,##STR25##

This intermediate can be synthesized by reduction of lower alkyl estersof 2-oximino-benzoylacetic acid using zinc dust in the presence ofacetic acid. The zinc dust (3.85-fold excess) is added gradually to theoxime maintaining a temperature between 45°-50° C. After completeaddition of the zinc dust the reaction is stirred for an additionalthree hours and then filtered to remove the zinc acetate. The product isisolated by concentration of the acetic acid solution.

EXAMPLE 16 Synthesis of the Semicarbazone of Methyl Benzoylglycine

The synthesis is useful when X and Y in Formula III are bound as═NNHC(:O)NH₂, ═NNHTs, ═NNHC(:O)R, or ═N--OH.

A 2M aqueous solution of semicarbazide hydrochloride containing 50mmoles of semicarbazide hydrochloride is added to 50 mmoles ofbenzoylglycine hydrochloride. Two equivalents of pyridine are added andthe solution is warmed gently until the product begins to crystallize.After two hours at room temperature the product is collected byfiltration. The semicarbazone of benzoylglycine is dissolved in ethylether and a solution, containing one equivalent of1-methyl-3-p-tolyltriazene, is added slowly. The product is isolated bywashing the ethereal solution rapidly with hydrochloric acid, then withaqueous sodium bicarbonate and finally dried and concentrated.

EXAMPLE 17 Syntheses of the Ethylene Dithioketal of MethylBenzoylglycinate

The synthesis is useful when X and Y are connected to C³ as --SCH₂ CH₂S--, or either is -SEt.

Methyl benzoylglycinate hydrochloride is dissolved in an acetic acidsolution containing two equivalents of ethanedithiol. The reactionmixture is heated to 60° C. and treated with three equivalents of borontrifluoride etherate. After heating for three hours the reaction mixtureis left to cool at room temperature overnight. The product, whichcrystallizes on standing, is isolated by filtration. A similar procedureusing two equivalents of ethanethiol is used to prepare the diethylthiolketal.

EXAMPLE 18 Syntheses of the Methyl Carbamate of Methyl Phenylserine

The following synthesis is useful when Y is H and X is --OC(:O)NHMe.

The amino moiety of the methyl phenylserinate is protected as thetrifluoroacetatmide by treating the free amine with one equivalent ofethyl trifluoroacetate. The N-protected methyl phenylserinate is treatedwith methyl isocyanate to form the corresponding methyl carbamate. Thetrifluoroacetyl group is removed by treatment with aqueous sodiumbicarbonate solution.

EXAMPLE 19 Syntheses of O-Methoxy Carbonyl Derivative of MethylPhenylserinate

The following synthesis is useful when Y is H and X is --O--C(:O)ME.

The amino moiety of methyl phenylserinate is protected by treatment withethyl trifluoroacetate to give the corresponding trifluoroacetamidederivative. The N-protected methyl phenylserinate is dissolved intetrahydrofuran and treated with methyl chloroformate to form thecorresponding O-methoxycarbonyl derivative of methyl phenylserinate. Thetrifluoroacetyl protecting group is removed by treatment with dilutesodium bicarbonate solution.

EXAMPLE 20 Synthesis of the Xanthate of Methyl Phenylserinate

The following synthesis is useful when Y is H and X is --O--C(:S)--SMe.

The amino moiety of the methyl phenylserinate is protected as theN-t-butoxycarbonyl derivative (BOC) by treating methyl phenylserinatewith di-t-butyl dicarbonate in the presence of aqueous sodiumbicarbonate. The N-protected methyl phenylserinate is then treated withcarbon disulfide and sodium hydroxide to form the corresponding sodiumxanthate which is then alkylated directly using methyl iodide. The BOCprotecting group is removed by treatment with trifluoroacetic acid.

EXAMPLE 21 Synthesis of the Methyl Ether of Methyl Phenylserinate

The following synthesis is useful when Y is H and X is --OMe.

The amino group of the methyl phenylserinate is protected as theN-t-butoxycarbonyl derivative which can be synthesized as describedabove. The N-protected methyl phenylserinate is dissolved intetrahydrofuran at 0° C. and treated with one equivalent of sodiumhydride. The resulting alkoxide is alkylated using two equivalents ofmethyl iodide. The BOC protecting group is removed using trifluoroaceticacid and the product is isolated in the usual manner.

EXAMPLE 22 Synthesis of the Dimethyl Ketal of Methyl BenzoylglycinateHydrochloride

The following synthesis is useful when X and/or Y is --OMe.

The dimethyl ketal of methyl benzoylglycinate hydrochloride can beprepared by simply dissolving the amino esther in methanol containing 3Amolecular sieves and stirring for 24 hours at room temperature. Themolecular sieves are removed by filtration and the product isolated byconcentration of the methanol.

EXAMPLE 23 Synthesis of the Methyl Sulfide of Methyl Phenylserinate

The following synthesis is useful when Y is H and X is --SMe.

Phenylpyruvic acid is treated with a strong base to generate thecorresponding ketone enolate. The enolate is treated with dimethyldisulfide to form the alpha sulfide which is neutralized and treatedwith hydroxylamine hydrochloride to produce the oxime. The oxime isreadily reduced using a mixture of zinc dust and acetic acid and thecarboxylic acid is fully esterified using 3-methyl-1-p-tolyl-triazene.

Note: L-threo-N-acetyl-beta-chlorophenylalanine ethyl ester (i.e., wherethe amine group is protected by acetyl, Y is H, and X is --Cl) may beprepared as shown in Volger, Helv. Chim. Acta, 33, Fasc. 7, No. 264, pp.2111-2117 (1950).

Procedures for isolation and recovery of the products made in theforegoing Examples 15-23 are routine. In occasional difficultisolations, resort can be had to standard chromatographic methods.

EXAMPLE 24 Prepartation of Beta-Chloro-phenylalanine Ethyl EsterHydrochloride ##STR26##

In a 50-ml round bottom flask fit with magnetic stirrer, refluxcondenser, and drying tube was placed 2.46 g. (10 moles) ofbetaphenylserine ethyl ester hydrochloride. The resulting suspension wasstirred and heated under reflux until all solid passed into solution(about 2.5-3 hours). The solution was cooled and the reflux condenserwas replaced by a still head, and the excess thionyl chloride wasremoved by distillation. The yellow orange residual oil was thenstripped of remaining volatiles at 50° C. under vacuum. The oil was thendissolved in 25 ml of anhydrous ether and allowed to crystallize at0°-5° C. The product was collected by filtration and washed with 10 mlof anhydrous ehter. The pure white solid amounted to 1.78 g. (67.4%yield. as a 1.9 to 1 mixture of erythro and threo isomers (by NMR).

Of the foregoing, Examples 1-14 and 24 are based on actual laboratorywork; Examples 15-23 are hypothetical but believed to work substantiallyas stated.

Resolution of (SS)- and (RR)- Isomers of Erythro-beta-phenylserinemethyl ester

In one embodiment the invention is operable to separate and recover the2 mirror image isomers of erythro-beta-phenylserine methyl ester. Theprocess schema is: ##STR27##

Blocked aspartic acid is coupled with erythro-phenylserine methyl esteras in Example 7. Only the (S,S)- (i.e., L-) isomer of the erythrocompound reacts. The reaction product mix thus contains Dipeptide I andfree unreacted (RR)- (or D-) erythro isomer. The reaction mix extractedwith ethyl acetate, from which Q-Asp-L-PhSerOMe, Dipeptide I, can berecovered as the hydrochloride. The free (SS)- or L- isomer is obtainedby treatment with thermolysin in water. this time, however, the enzymefunctions as a hydrolyzing agent, giving back the blocked aspartic acidplus free L-(S,S)-PhSerOMe. To separate the two, HCl is added and theacidified solution is extracted with ethyl acetate to recover Q-Asp. TheL-PhSerOMe stays in the acidified solution. The blocked aspartic acid isrecycled.

The 2 optical isomers if erythro-beta-phenylserine have uses principallyin the pharmaceutical field,

In the above schema, other amino acids, such as Q-phenylalanine can beused in lieu of Q-Asp, and racemates other than D,L-PhSer can be usede.g., Formula II in the D,L-form.

Some Variations

Supports for immobilized enzymes are well-known in the art. They includepolyacrylate resins, porous glass beads, hydrophilic gels, vermiculite,and the like.

Suitable organic solvents for the reaction of the 2 substrates (e.g.,PhSerOMe and Q-Asp) include a lower alkyl halide such as chloroformm orethylene dichloride; an ester of carboxylic acid, such as ethyl acetate,isopropyl acetate, butyl acetate, and isobutyl acetate; a ketone such asmethylisobutyl ketone; and an aromatic hydrocarbon such as benzene,toluene, or a mixture. Other suitable solvents include butanediol,glycerol dimethylformamide, dimethylsulfoxide, triethylene glycol,acetonitrile, methanol, ethanol, t-butanol, cyclohexanol, dioxane,isopropyl ether, trichlorethylene, tetrachloroethylene, and the like.

In a preferred embodiment protease-catalyzed peptide synthesis iscarried out in the presence of water-miscible organic solvent to improvethe solubility of reactants and to suppress the ionization of thereacting carboxyl group, resulting in a shift of the equilibrium towardssynthesis. Water-miscible organic solvents that can be used arebutanediol, glycerol, dimethylformamide, dimethylsulfoxide, triethyleneglycol, acetonitrile, and the like. Examples: trypsin in 50%dimethylformamide--see J. Amer. Chem. Soc. 101, 751 (1979); 33%dimethylformamide--see J. Biol. Chem., 255, 8234 (1980); papain inethanol-buffer solution--see Biochem. Biophys. Res. Commun. 91, 693(1979); and prolysin from B. subtilis var. amyloliquefaciens in 15%methanol or dioxane--see Bull. Chem. Soc. Japan, 51, 271 (1978).

In a biphasic system the enzyme and substrates are dissolved in theaqueous, buffered solution and the product diffuses to the nonpolarorganic phase such as benzene, toluene, dichloroethane,tetrachloroethylene, and the like.

Each of the two reactants (Formula II and Formula III, e.g., Q-Asp andPhSerOMe) can be used in a concentration of about 0.01 molar to 1.5molar, and preferably about 0.1 to 0.5 molar. The mole ration ofQ-Asp:PhSer suitably ranges between 10:1 and 1:10, and preferablybetween 1:1 and 1:5.

In the catalytic hydrogenation of a dipeptide in the Dipeptide I Classto one in the Hydroxy-Aspartame Class (Formula XI), the hydrogenpressure can be in the range of atmospheric pressure to 1,500 psig andthe temperature 0°-150° C. The same pressure and temperature rangesapply in the hydrogenation of dipeptides in Formula XI to aspartame orits hohologs, and in the hydrogenation of dipeptides in Dipeptide IClass (Formula X) direct to aspartame or ester homolog. In thesehydrogenation the catalyst is suitably Pt or PtO₂ ; or Pd, Pd black, orPd(OH)₂. The support can be carbon, barium sulfate, alumina, or thelike. As catalysts, Raney Ni and Raney CO are also useful.

In the coupling reaction, a catalytic amount of enzyme is used,typically 10 mg-3 g of enzyme (dry basis) per millimole of aspartic acidcompound in a continuous reactor. In batch runs, the ratio is suitably10-150 mg of enzyme (dry basis) per millimole of aspartic acid ofFormula II, e.g., Q-Asp. As will be evident, in a continuous reactor, atany given point in time there is a considerable amount of enzyme in thereactor column in proportion to aspartic acid compound. In a batchprocess this is of course not the case.

The temperature for the coupling reaction is suitable in the range20°-70° C., and preferable 30°-50° C. The reaction is generallysubstantially complete in 2-10 hours. If the reaction is undulyprolonged after it is complete, some decompositions may result, withformation of benzaldehyde.

Some Further Consideration of Nonobviousness

Although the addition of a substituent at the beta position of PheOMemay appear as a minor chemical range, these derivatives behavecompletely differently from the unsubstituted compound when analyzed assubstrates for proteases. For instance, while L-PheOMe is hydrolyzed toL-PheOH by chymotrypsin, papain, and pronase, erythro- andthreo-PhSerOMe were not hydrolyzed when these proteases were tried.

The utilization of proteases as catalysts in the sythesis of peptides iswell established. This work, however, was involved by and large with thecondensation of the common naturally occuring amino acids. So far as isknown, the instant invention is the first time that derivatives of therare amino acid, phenylserine, have been employed in protease-catalyzedpeptide synthesis.

Proteases are known to be stereospecific, i.e., the catalyze thespecific condensation of the L-isomers, leaving the D-isomers intact.One might expect that the presence of another asymmetric center as inPhSerOMe, however, in a more remote site from the reaction site(beta-position) will not cause any difference in stereoselectivity. Theresults, however, show that thermolysin is sensitive to thestereochemistry at C³ in compounds of Formula III and in the case ofphenylserine methyl ester only the L-erythro isomer is utilized in thecondensation reaction.

We claim:
 1. Process for producing a dipeptide of the formula ##STR28##wherein B represents hydrogen or Q; Q represents an amino acidprotective group; V represents hydrogen or an alkyl group having 1, 2,3, or 4 carbon atoms; C¹ and C² have the common natural configuration ofnaturally occurring amino acids; and Y is hydrogen and X is chloro orhydroxyl,said process comprising: reacting B-substituted aspartic acidof the formula ##STR29## wherein X, Y, and V are as defined above andthe C² carbon has the common natural configuration of naturallyoccurring amino acids; said reaction being conducted in(d) awater-immiscible solvent in the presence of a metallo-proteinase, (e) awater-miscible solvent in the presence of a non-metallo-proteinase, or(f) a water-immiscible solvent in the presence of anon-metallo-proteinase.
 2. Process according to claim 1 in which theproteinase is immobilized.
 3. Process according to claim 1 in which theproteinase is thermolysin, trypsin, pronase, prolysin, papain,collagenase, or crotulus atrox protease.
 4. Process according to claim 1in which Q is carbobenzoxy, p-methoxybenzyloxycarbonyl,t-butoxycarbonyl, phenylacetyl, acetoacetyl, N-benzylidene, benzoyl,benzyl, t-amyloxycarbonyl; chloroacetyl, carbamyl,3,5-dimethoxybenzyloxycarbonyl; 2,4,6-trimethylbenzyloxycarbonyl;p-phenylazobenzyloxycarbonyl; p-toluenesulfonyl; o-nitrophenylsulfonyl;or trifluoroacetyl.
 5. Process according to claim 2 in which B and Y areH and X is --OH, thereby to make hydroxy-aspartame.
 6. Process accordingto claim 2 in which B is Q and the mole ratio of the Q-protectedaspartic acid to the second amino acid is 10:1 to 1:10.
 7. Processaccording to claim 1 in which 10-3000 mg of proteinase is used permillimole of Q-protected aspartic acid.
 8. A process for producing adipeptide of the formula ##STR30## comprising treating an amino acidester having the formula ##STR31## or a salt thereof, to replace X and Ywith H, to provide a phenylalanine ester having the formula, ##STR32##and reacting said phenylalanine ester with B-substituted aspartic acidof the formula ##STR33## or a salt thereof; said reaction beingconducted in (a) a water-immiscible solvent in the presence of ametallo-proteinase,(b) a water-miscible solvent in the presence of anon-metallo-proteinase, or (c) a water-immiscible solvent in thepresence of a non-metallo-proteinase;wherein B represents hydrogen or Q;Q represents an amino acid protective group; V represents hydrogen or analkyl group having 1, 2, 3, or 4 carbon atoms; C¹ and C² have the commonnatural configuration of naturally occurring amino acids; and (d) Y ishydrogen and X is chloro or hydroxyl.
 9. Process according to claim 1 ofmaking a dipeptide comprising(I) in a first reaction, reactingB-substituted aspartic acid of the formula ##STR34## or a salt thereofwith a second amino acid having the formula ##STR35## thereby to form acompound of the formula ##STR36## and (II) treating said latter compoundto replace X and/or Y with H; or, when B is Q, to replace Q withH;wherein B represents hydrogen or Q; Q represents an amino acidprotective group; V represents hydrogen or an alkyl group having 1, 2,3, or 4 carbon atoms; C¹ and C² have the common natural configuration ofnaturally occurring amino acids; said first reaction being conducted in(d) a water-immiscible solvent in the presence of a metallo-proteinase,(e) a water-miscible solvent in the presence of anon-metallo-proteinase, or (f) a water-immiscible solvent in thepresence of a non-metallo-proteinase.
 10. Process according to claim 9in which the solvent is chloroform, ethylene dichloride, ethyl acetate,isopropyl acetate, butyl acetate, isobutylacetate, methylisobutylketone, benzene, toluene, butanediol, glycerol, dimethylformamide,dimethylsulfoxide, triethylene glycol, acetonitrile, ethanol, methanol,dioxane, isopropylether, tetrachloroethylene, trichlorethylene,t-butanol, or cyclohexanol.
 11. Process according to claim 9 in which Qis carbobenzoxy, p-methoxybenzyloxycarbonyl, t-butoxycarbonyl,phenylacetyl, acetoacetyl, N-benzylidene, benzoyl, benzyl,t-amyloxycarbonyl; 3,5-dimethoxybenzyloxycarbonyl;2,4,6-trimethylbenzyloxycarbonyl; p-phenylazobenzyloxycarbonyl;p-toluenesulfonyl; o-nitrophenylsulfonyl, trifluoroacetyl, chloroacetyl,or carbamyl.
 12. Process according to claim 9 in which the compoundresulting from catalytic hydrogenation in (II) is hydroxy-aspartame. 13.Process according to claim 9 in which the hydrogenation is conducted at0°-150° C., at a hydrogen pressure ranging from atmospheric to 1500psig, in the presence of a catalyst of Pt, PtO₂, Pd, Pd black, orPd(OH)₂ on carbon, barium sulfate, or alumina; or in the presence of acatalyst of Raney Ni or Raney Co.
 14. Process according to claim 13 inwhich the hydrogenation is conducted at a temperature in the range ofroom temperature to 45° C., at a hydrogen pressure ranging from 45 to 60psig, in the presence of Pd(OH)₂ catalyst.
 15. Process according toclaim 9 in which first one and then the other of X and Y is replacedwith hydrogen.
 16. Process according to claim 9 in which B is Q, and Q,but not X or Y, is replaced with H.
 17. A method according to claim 1for manufacturing a dipeptide from an N-substituted aspartic acid and(S,S)-beta-phenylserine lower alkyl ester, said method comprisingsubjecting said N-substituted aspartic acid and said phenylserine loweralkyl ester to a reaction in an organic solvent immiscible with water inthe presence of a water-containing immobilized metallo-proteinase. 18.Method according to claim 17 in which dipeptide product isN-carbobenzoxy-L-alpha-aspartyl-L-erythro-beta-phenylserine methyl esteror N-butoxycarbonyl-L-alpha-aspartyl-L-erythro-beta-phenylserine methylester.
 19. The method according to claim 9 of preparing a dipeptide,being L,L-aspartame or its lower alkyl ester homolog, comprising(A)subjecting Q-substituted aspartic acid and beta-phenylserine lower alkylester, said ester containing (2S,3S) isomer, to reaction in an organicsolvent immiscible with water in the presence of a water-containingimmobilized metallo-proteinase, thereby to form a first dipeptide of theformula ##STR37## (B) treating said first dipeptide to replace Q with Hand thereby forming a second dipeptide of the formula ##STR38## and (C)catalytically hydrogenating said second dipeptide thereby formingaspartame or its lower alkyl ester homolog.